Method for creating a leukocyte rich sample from a mixed population of blood cells

ABSTRACT

A method and apparatus for creating a leukocyte rich sample from a mixed population of blood cells. The method comprises the steps of: separating an amount of blood into layers that include a buffy coat layer having a mixed population of blood cells including plasma and red blood cells; providing a chamber having an opening for receiving a fluid; causing the buffy coat layer to enter the chamber; centrifuging the chamber to separate the buffy coat layer into at least a plasma containing layer, a leukocyte containing layer, and a red blood cell containing layer; and separating the leukocyte containing layer from the other layers in the chamber.

This application is a continuation application under 37 C.F.R. §1.62 ofprior application Ser. No. 08/434,623, filed on May 4, 1995, abandoned,which is a division of application Ser. No. 08/164,517, filed on Dec. 9,1993, U.S. Pat. No. 5,686,283 which is a continuation-in-part of U.S.Ser. No. 08/037,525 filed Mar. 24, 1993, U.S. Pat. No. 5,498,520, whichis a continuation of U.S. Ser. No. 07/833,285 filed Feb. 10, 1992 nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to diagnostic techniques. Morespecifically, the present invention relates to the testing of bloodunits for viral contamination.

In a variety of therapies, such as transfusion and transplants, bodyfluids, especially blood components, such as red blood cells, platelets,plasma, and bone marrow, are infused from one or more individuals into apatient. Although such therapies provide treatments, some of which arelife saving, that cannot otherwise be provided, due to the transmissionof infectious diseases there may be potential risks to such therapies.

For example, it is known that blood can carry infectious agents, such ashepatitis virus, human immunodeficiency virus (an etiological agent forAIDS), cytomegalovirus, Epstein Barr virus, and herpes virus. Althoughscreening methods exist to identify blood that may include such viruses,current screening methods do not assure that every blood unit thatcontains such a virus is identified.

In this regard, one of the difficulties in testing blood components forviral contamination, such as HIV, is that many current diagnostic testsare based on an identification of antibodies. Accordingly, they willonly exhibit a positive test result if the blood unit includesantibodies for the virus, e.g., anti-HIV. With respect to intracellularviral infections, an individual, however, does not generate antibodiesimmediately upon infection. Rather there is a "window period" thatextends from the initial infection of the patient with a virus to thegeneration of antibodies. When an individual is in this window period,diagnostic tests that are based on antibodies will not identify theindividual, or the blood unit, as being infected. But, even thoughantibodies are not present, the blood unit can still transmit aninfection.

It is believed that this window period, with respect to HIV infection,extends from approximately six weeks to 48 months. During this timeperiod, an individual who has been infected with HIV and accordingly,whose blood will transmit same, will register a negative antibodyresponse. Therefore, current screening methods will not identify asviral contaminated a blood unit from an individual who is infected withHIV but who has not generated anti-HIV.

In order to identify blood units that may be contaminated because anindividual is within the window period, recent attempts have focussed onthe use of nucleic acid sequencing diagnostic techniques.

Specifically, attempts have been made to use polymerase chain reaction(PCR) techniques for detecting nucleotide sequences for HIV virus.

A number of methods of using PCR are known.

Briefly, in one PCR method, a sample containing DNA is placed in areaction tube including appropriate buffers, nucleoside triphosphates, athermostable DNA polymerase and oligonucleotide primers complementary tothe ends of a region of DNA of interest. Initially, to denature thedouble-stranded DNA under study, the temperature of the reaction israpidly increased. The temperature is then decreased allowing theoligonucleotide primers to anneal to their complementary sequences. Byincreasing the temperature, a DNA extension occurs at the optimaltemperature of activity for the polymerase. By repeating these cycles ofdenaturation-annealing-extension, a single sequence of a few hundredbase pairs can be amplified. This amplification can be in the range of afactor of 10⁶ and detected with relative ease. Conway, "Detection ofHIV-1 by PCR in Clinical Specimens", "Techniques in HIV Research",Stockton Press (1990).

Although nucleic acid sequencing techniques are very sensitive, they arealso sample specific. Moreover, the samples of the blood units so testedare rendered unusable for therapeutic applications. Because of this,currently nucleic acid sequencing diagnostic methods do not provide amethod for insuring that blood units do not have viral contamination.

For example, if one were to attempt to use a PCR technique to test asample of a blood unit using standard technology, one could not insurethat the unit did not contain viral contamination even if the test wasnegative. In this regard, because a PCR test is sample specific unlessthe whole blood unit was tested, it would not accurately reflect thatthe unit did not include viral contamination; if only a 10 ml samplewere tested, the remaining 290 ml of a 300 ml could contain a viralcontaminant.

A PCR test will only determine if there is a viral agent in the samplebeing assayed, not in the entire unit. Because the test destroys theviability of the component, heretofore, it was not possible to test theentire component to ensure that it is not contaminated with, forexample, HIV.

Accordingly, there is a need for improved diagnostic testing of bloodunits to determine viral contamination during the "window period" ofcontamination.

SUMMARY OF THE INVENTION

The present invention provides a method for testing a blood unit forviral contamination that does not render the blood unit unusable fortherapeutic applications. The method allows one to effectively samplethe entire blood unit using, for example, nucleic acid sequencingtechniques. The method improves the accuracy of diagnostic tests forviral contaminants.

Although typically to increase the accuracy of a diagnostic test forinfectious disease it is desirable to increase the sample volume of thematerial being assayed, with respect to blood components this is notpossible. In the case of blood and its components, only a small fractionof the total volume of the blood component can be sacrificed forperformance of diagnostic assays for infectious diseases. The inabilityto sample the whole blood unit places a finite limit on the accuracy ofdiagnostic testing (including nucleic acid amplification technology) dueto sampling volume. In this regard; 1) the assay result can be a truenegative, but the unit positive; or 2) the assay result can be a falsenegative due to lack of sensitivity of the assay.

The present invention addresses both of these limitations.

To this end, the present invention provides a method for testing a bloodunit for viral contamination without rendering the blood unit unusablefor therapeutic applications. The method comprises the steps of:removing and collecting from a blood unit a majority of the leukocytespresent therein; and using the collected leukocytes to test the bloodunit for viral contamination.

In an embodiment, the present invention provides a method for testing ablood unit for viral contamination without rendering the blood unitunusable for therapeutic applications comprising the steps of separatingthe blood unit into a plasma rich platelet component, a buffy layer, anda red blood cell component; removing the buffy layer; and assaying thebuffy layer for viral contamination.

In a preferred embodiment, the present invention provides a method forcreating a leukocyte rich sample from a mixed population of blood cellscomprising the steps of separating an amount of blood into layers thatinclude a buffy coat layer having a mixed population of blood cellsincluding plasma and red blood cells; providing a chamber having anopening for receiving a fluid; causing the buffy coat layer to enter thechamber; centrifuging the chamber to separate the buffy coat layer intoat least a plasma layer, a leukocyte layer, and a red blood cell layer;and separating the leukocyte layer from the other layers in the chamber.

In an embodiment, the leukocyte layer includes less than or equal to5×10⁸ red blood cells.

In an embodiment, the leukocyte layer includes less than or equal to5×10⁹ platelets.

In an embodiment, the chamber is exposed to a centrifugational force ofat least 300×g.

In an embodiment, the chamber is exposed to a centrifugational force ofapproximately 750×g.

In an embodiment, the chamber is centrifuged for approximately 5 toabout 10 minutes.

In an embodiment, the chamber is defined by a syringe.

The present invention, in another embodiment, provides a method fortesting a blood unit for viral contamination without rendering at leasta platelet rich plasma layer and a red blood cell rich layer of theblood unit unusable for therapeutic applications comprising the stepsof: separating an amount of blood into a platelet rich plasma layer, ared blood cell rich layer, and a buffy layer that includes a mixedpopulation of blood cells including plasma and red blood cells;providing a chamber having an opening for receiving a fluid; causing thebuffy coat layer to enter the chamber; centrifuging the chamber toseparate the buffy coat layer into at least a plasma layer, a leukocytelayer, and a red blood cell layer; separating the leukocyte layer fromthe other layers in the chamber; assaying the leukocyte layer todetermine viral contamination; and if the assay determines no viralcontamination is present in the leukocyte layer using at least one ofthe platelet rich plasma layers or red blood cell rich layer fortherapeutic applications.

In another embodiment, a method for testing a blood unit for viralcontamination without rendering at least either a platelet rich plasmalayer or a red blood cell rich layer of the blood unit unusable isprovided comprising the steps of: separating an amount of blood intolayers that include a buffy coat layer having a mixed population ofblood cells including plasma and red blood cells; providing a syringehaving an opening for receiving a fluid, a chamber, and a plungerreceived within the chamber; causing the buffy coat layer to enter thechamber; securing means for preventing axial movement of the plungerinto the chamber around at least a portion of the plunger; centrifugingthe syringe to separate the buffy coat layer into at least a plasmalayer, a leukocyte layer, and a red blood cell layer; and separating theleukocyte layer from the other layers in the chamber.

Additionally, the present invention provides a system for use inobtaining a leukocyte rich sample from a mixed population of bloodcells. The system includes a syringe including a chamber having a firstopening for receiving a fluid and a plunger received within the chamberfor causing fluid to be urged into and out of the chamber. The systemalso includes removable means that prevents the plunger from axialmovement into the chamber. In an embodiment, the means is a sleevecoupled around at least a portion of the plunger. In an embodiment, thesyringe includes a cannula extending from the opening.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the detailed description of thepresently preferred embodiments and from the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically an embodiment of the method of thepresent invention for testing a blood component for viral contamination.

FIG. 2 illustrates schematically a further embodiment of the method ofthe present invention for testing a blood component for viralcontamination.

FIG. 3 illustrates an embodiment of the modified syringe of the presentinvention.

FIG. 4 illustrates a cross-sectional top view of the embodiment of thesyringe of FIG. 3 taken along lines IV--IV.

FIGS. 5a-5d illustrate, schematically, an embodiment of the process ofthe present invention.

FIG. 6 illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of slow speedbuffy coat preparations at 1000×g with an embodiment of the syringe ofthe present invention.

FIG. 7 illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of slow speedbuffy coat preparations at 750×g with an embodiment of the syringe ofthe present invention.

FIG. 8 illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of slow speedbuffy coat preparations at 500×g with an embodiment of the syringe ofthe present invention.

FIG. 9a illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of buffy coatpreparations at different centrifugation speeds with an embodiment ofthe syringe of the present invention for fresh blood, i.e., less than 4hours old.

FIG. 9b illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of buffy coatpreparations at different centrifugation speeds with an embodiment ofthe syringe of the present invention for a 48 hour old buffy coat.

FIG. 10a illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of buffy coatpreparations at different centrifugation speeds with an embodiment ofthe syringe of the present invention for fresh blood, i.e., less than 4hours old.

FIG. 10b illustrates graphically test results from Example No. 1 andspecifically, phenotype of cells pre and post processing of buffy coatpreparations at different centrifugation speeds with an embodiment ofthe syringe of the present invention for a 24 hour old buffy coat.

FIG. 11 illustrates schematically the use of an embodiment of thepresent invention for validating virus depletion/inactivation process.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The present invention provides an improved method for testing a bloodcomponent for a viral contaminant. The method of the present inventionimproves the accuracy of diagnostic tests without sacrificing the bloodunit to be administered. Additionally, the present invention provides anapparatus and improved system for testing blood compounds. To this end,the present invention allows the entire unit of whole blood, red bloodcells, or platelet concentrate to be sampled for viral contaminationwithout rendering the cell product unusable for therapeuticapplications.

In an embodiment, the invention includes the steps of using a modifiedsyringe to collect a buffy coat fraction from a whole blood separationprocess that separates blood into layers including a buffy coat layerthat has a mixed population of leukocytes, red blood cells, andplatelets. After the syringe has received the buffy coat layer, meansfor preventing the plunger from moving into the syringe chamber areattached to the syringe. The syringe is centrifuged. Due to the meansfor preventing, the plunger does not move into the syringe chamber,preventing the buffy coat layer from being expelled from the syringe.

The buffy coat through the centrifuge process is separated into plasma,leukocytes (white blood cells) and red blood cells. The plasma can thenbe expressed off the leukocytes and the leukocytes can be easilysegregated for testing. Thus, a rich leukocyte sample is capturedwithout damaging the blood components. The methods and devices for socapturing the leukocytes will be set forth in detail hereinafter.

There are three principal indications for the transfusion of a bloodproduct: 1) deficiency in oxygen-carrying red blood cells; 2) deficiencyin hematologic factors related to blood clotting, includes platelets orprotein coagulation factors; and 3) deficiency in plasma volume.Patients requiring a transfusion do not receive whole blood but thespecific component required to overcome the clinical deficiency. Forexample, patients undergoing chemotherapy or radiation therapy requireprimarily platelets and to a lesser degree red cells. Bone marrow orother organ transplant and dialysis patients generally require only redblood cells.

Leukocytes are unwanted because they are not relevant to the therapeuticeffects of oxygen-carrying red blood cells, platelet or plasma, and havebeen implicated as increasing the risks associated with bloodtransfusion for their role in alloimmunization to HLA antigens and posttransfusion infection by acting as virus carrying cells. Typically, theleukocytes are removed by a filtering process in approximately 10% ofthe blood components that are transfused.

When a filtering process is used, the leukocytes captured in thesefilters are discarded. As set forth in detail below, in an embodiment ofthe invention, the filters containing the leukocytes can be used as astarting material for a process to recover and/or amplify viral markers(viral nucleic acid sequences and/or antigens).

Pursuant to an embodiment of the present invention, a majority, greaterthan 50%, of the leukocytes are collected. Depending on the method used,up to 99.8% of the leukocytes may be collected. Leukocytes typicallycomprise 0.25% of the cellular composition of blood. A leukocyte countof 7.5 million/ml of blood is considered average for an adult. In a 500ml unit of whole blood, containing 63 ml of anticoagulant solution,3.2×10⁹ leukocyte cells will be present.

Because the leukocytes are a source of infectivity in the blood, theyprovide a desirable material for diagnostic assays. Furthermore, becausethe collected leukocytes are derived from the entire unit of bloodcomponent to be transfused, as opposed to a small segment thereof, animproved diagnostic procedure is achieved.

FIG. 1 illustrates schematically an embodiment of the process of thepresent invention. The blood component (which can be whole blood, redcell concentrate, platelet concentrate, or pooled platelet concentrate)is first passed through a filter which is designed to capture 90 togreater than 99% of the leukocytes present.

Leukocyte filters that can be used pursuant to the present inventioninclude the Sepacell R-500 filter available from Asahi Corp., Tokyo,Japan and the RC-100 for blood filtration and PL-100 for plateletfiltration, both available from Pall Biomedical Corp., East Hills, N.Y.It has been reported in Biotechnology & Medical, Aug. 19, 1988 that anAsahi filter is able to remove 99.8% of the leukocytes present in wholeblood.

The filtered blood component is collected in a blood collection bag. Theblood component is stored pending the results of the diagnostic test.

Pursuant to an embodiment of the present invention, the leukocyte filteris utilized as a source material for diagnostic tests for infectiousdisease antigen and nucleic acid sequences. To this end, the filter istreated to lyse the cells contained in the filter. This allows therecovery of nucleic acids and antigens that are contained on and withinthe leukocytes.

The cells are lyse by using a lysing reagent. The lysing reagentpreferably will be a solution containing a detergent to break thecytoplasmic membrane and release the nuclei of the cells.

The cell lysate is then flushed out of the filter using an isotonicsaline solution and collected. This resultant cell lysate is then usedas a test material for diagnostic assays (nucleic acid amplificationtechnology for viral nucleic acid sequences, e.g., PCR or 3SR or viralantigens, e.g., enzyme-linked immunoadsorbent assays).

Because the assay, pursuant to the present invention, will be based onan analysis of the great majority of the leukocytes present in the bloodunit, even when using sample specific analysis such as PCR methods onecan be assured that a negative result is accurate even if an individualis in a window period of infection.

Once a negative result is obtained using the cell lysate, the bloodcomponent contained in the collection container can then be transfusedinto a patient.

FIG. 2 illustrates schematically another embodiment of the presentinvention. If necessary to increase assay sensitivity (particularly forantigen capture assay systems), the leukocyte filter can be utilized asa culture chamber for viral replication.

In this case, again a blood component (whole blood, red cellconcentrate, platelet concentrate, or pooled platelet concentrate) isfiltered through a leukofilter. The leukofilter is separated from theblood bag container, containing the filtered blood component, and thefilter is flushed with tissue culture media such as 10% Fetal Calf Serumin RPMI 1640 media. In some cases, it may be desirable to supplement themedia with cytokines (e.g., IL-2 or T-cell mitogen (e.g., PHA)) topromote viral replication in the leukocytes and to sustain the viabilityof the cells in the filter.

In addition, in some cases, it may be desirable if the tissue culturemedia is supplemented with viral host cell lines (example H9 cells forHIV). In this case, the filter would be used as a culture chamber forthe leukocytes captured from the blood and for virus host cells. The H9cells can then be infected by HIV present in the captured leukocytes andcan serve to amplify the production of viral antigen (e.g., HIV p24antigen).

After the appropriate culture period of approximately 1 to 5 days, thefilter can be treated to accomplish lysis of the trapped cells by one ofseveral means (freeze-thaw, detergent, hypotonic shock). The resultingcell lysate can then be collected by flushing the filter with anisotonic saline solution.

The collected cell lysate can be tested using conventional antigencapture assay systems such as, for example, DuPont HIV p24 Care Profile,Elisa Kit or Coulter HIV p24 Antigen Assay (Hialeah, Fla.) for the HIVp24 antigen or by nucleic acid amplification methodologies, such as GeneAmplimer HIV-1 Reagents with GeneAmp® PCR Core Reagents and Perkin-ElmerCetus DNA Thermal Cycler, available from Perkin-Elmer Cetus. In somecases, the cell lysis may not be required due to release of the virusinto the tissue culture media from infected cells.

Again, by using this method, even if a sample specific diagnostic testis used, because the test is with respect to the great majority ofleukocytes present, a negative test will ensure that the blood componentis not contaminated. The blood component contained in the blood bagcontainer can then be transfused into a patient.

In an embodiment of the method of the present invention, instead offiltering the blood component to capture the leukocytes, acentrifugation process that separates a whole blood unit into variouscomponents can be used. During a typical centrifugation process, thewhole blood is separated into a top portion including platelet richplasma, a middle or buffy coat layer containing white cells andleukocytes, and a bottom red blood cell rich layer. Typically, the buffycoat layer is discarded.

Pursuant to the present invention, the buffy coat layer is used fordiagnostic applications. To this end, the buffy coat can be subjected tonucleic acid amplification, e.g., PCR or 3SR or viral antigen captureassay. By testing the buffy coat layer, if a negative test results, oneis assured that both the platelet rich plasma component and red bloodcell component are viral contaminant free. It is believed that bycollecting the buffy coat using standard techniques, at leastapproximately 80% of the leukocytes are recovered.

In an embodiment, the following method is used:

1. Centrifuge the blood bag in an inverted position in a refrigeratedcentrifuge containing whole blood at 5° C. using a "heavy" spin.

2. Hang the centrifuged, inverted bag on a ring stand or inverted plasmaexpresser. The temperature of the blood must not exceed 10° C. duringthe procedure. Allow the bag to hang undisturbed for several minutes.

3. Place the transfer bag on a scale (e.g., dietary scale) below theblood bag. Adjust the scale to zero.

4. Penetrate the closure of the primary bag, avoiding agitation of thecontents and allow red blood cells to flow to the transfer bag. At least80% of the red blood cells must be transferred to the satellite bag. Tocalculate the amount of red blood cells to be expressed estimate theamount of blood (excluding anticoagulant) in the bag multiplied by thedonor's hematocrit (assume 40% for females and 43% for males) (1 ml ofred blood cells (RBC) weighs 1.06 gm).

5. The remaining blood in the bag contains the buffy coat, some residualred blood cells, and the plasma.

6. The contents are mixed and the preparation is centrifuged to pelletthe buffy coat.

7. The plasma is expressed into a satellite container.

8. The residual red cells in the buffy coat can be lysed by adding ahypotonic ammonium chloride solution or other red cell lysing reagent togenerate a purified buffy coat preparation.

9. This cell preparation would be processed for PCR or other analysisusing standard methodologies.

In an embodiment, preferably, the buffy coat is collected within 24hours from the time of donation.

Other methods can be used to obtain the buffy coat, using a deviceavailable from subsidiaries of Baxter International sold under thetrademark Optipress® and using disposables sold under the trademarkOptipak®. Reference is also made to U.S. Pat. Nos. 4,350,585 and4,608,178, the disclosures of which are hereby incorporated herein byreference.

Using traditional separation methods, the resultant buffy coat layerthat is obtained has a mixed population. The layer thereby includes inaddition to leukocytes, platelets, and red blood cells. This additionalpopulation may interfere with a PCR analysis of the leukocytes that arepresent in the buffy coat. Pursuant to the present invention, a methodis provided for creating a substantially purified leukocyte sample froma traditionally obtained buffy coat layer that has a mixed population.

To this end, after a fraction of the buffy coat is collected throughtypical methods using, for example, an Optipress® device, a modifiedsyringe can be used to collect the buffy coat fraction. Referring now toFIG. 3, in an embodiment, the syringe 20 includes a body 25, an opening24 and a chamber 26. Preferably, as illustrated in FIG. 5, the syringeincludes a cannula 22. However, it is not necessary, in certainapplications, to provide a cannula on the syringe. Additionally,depending on the application or use, the cannula may be a pointedcannula or a blunt cannula.

Received within the chamber 26 is a plunger 28. The plunger 28 causesfluid to be sucked into the chamber 26 and expelled therefrom as isknown in the art.

Additionally, the modified syringe system 20 includes a plunger sleeve30. The plunger sleeve 30 prevents, as discussed in detail below, theplunger from moving into the chamber 26.

FIGS. 5a-5d illustrates schematically an embodiment of the method of thepresent invention. In use, the cannula 22 pierces a container 32including a buffy coat fraction 34 that includes a mixed population. Thecontainer can contain blood that has been separated into layersincluding a buffy coat layer, or the container 32 as illustrated canonly contain the buffy coat layer 34. The amount of buffy coat collectedcan vary depending on the component that was separated and the volume ofsame. Therefore, the volume of the chamber can, if desired, likewisevary.

Once the cannula 22 has pierced the container 32, the buffy coat layeris collected by pulling the plunger 28 out of the chamber 26. Thiscauses the buffy coat layer 34 to be received by the chamber 26 of thesyringe 20.

Pursuant to the present invention, after the buffy coat 34 has beenreceived within the syringe 20, and specifically, the chamber 26, theplunger sleeve 30 is located over the plunger 28. Although, in theillustrated embodiment, the plunger sleeve is a piece of plastic thatincludes a slit 31 so it that can be slipped over and coupled around theplunger 28, any means for preventing movement of the plunger can beutilized. For example, the plunger sleeve 20 could comprise a memberhaving a hinge allowing the sleeve to be opened and coupled around theplunger. Likewise, the plunger sleeve could be a multi-piece member thatis secured around the plunger. The plunger sleeve 30 prevents axialmovement of the plunger 28 into the chamber 26 of the syringe 20.

After the plunger sleeve 30 has been secured to the syringe 20, thesyringe 20 can then be centrifuged. To this end, preferably, the cannula22 is removed and the opening 24 is removably sealed with a cap or othermeans. As illustrated in FIG. 5c, the syringe 22 is then placed within acentrifuged tube 36. By way of example, if a 5 cc syringe is used, a 50cc centrifuge tube can be used to receive the syringe.

The syringe 22 is then centrifuged causing the buffy coat mixedpopulation to separate into a plasma layer 40, a leukocyte layer 42, anda plasma rich red blood cell layer 44. This separation is due to thedifferent densities of the cellular components in the buffy coat 34. Asillustrated in FIG. 5d, the heaviest of the cells, the erythrocytesbecome packed at the bottom of the chamber 26 with leukocytes andplatelets forming sequential layers within the chamber 26 of the syringe20. As illustrated in FIG. 5d, therefore, the plasma layer 40 will bethe first layer, the leukocyte layer the second layer, and the red bloodcell layer 44 the third or bottom layer.

After the centrifugation process, the leukocyte layer 42 is separatedfrom the remaining layers for PCR analysis. A variety of methods can beused to so separate the layers. In the illustrated embodiment, a samplecollection tubing 46 can be coupled around the opening 24 of the syringe20. The plunger sleeve 30 is removed, and the plunger 28 is then biasedinto the chamber causing first the plasma layer 40 to be expressedthrough the sample collection tube 46. This layer should besubstantially clear or have a slightly yellow color.

The leukocyte layer 42 will then be expressed into the sample tube 46after the plasma layer 40. The practitioner can easily determine whenthe leukocyte layer 42 is beginning to be received in the collectiontube 46 as it has a white color distinct from that of the plasma. Oncethe entire leukocyte layer 42 has been received within the sample tube46, the tube is then sealed. The practitioner can easily determine theline of demarcation between the leukocyte layer 42 (white in color) andthe red blood cell layer 48 (red in color). The tube 46 can be sealedthrough a variety of means including heat sealing.

The tube 46 containing the leukocytes then can be used for PCR analysis.If desired, for example, the leukocytes can be transferred to anothercontainer for PCR diagnostics or storage.

As an alternative to using a tube to collect the leukocytes, avacutainer tube (not illustrated) can be used. To this end, a smallersyringe sleeve (not illustrated) would be attached to the chamberplunger that would stop when a given volume of sample is collected whileusing the vacutainer tube to draw out the sample.

The method of the present invention allows the collection of a highconcentration of leukocytes in less than 15 minutes with minimumtechnique manipulation. Additionally, the syringe 20 and chamber 26 is aclosed system that will decrease exposure of the blood centertechnicians to blood borne pathogens. The collected leukocytes from thechamber 26 will be a substantially pure, rich collection of leukocytesthat can be used as a source of cells for screening the blood productsfor viral contaminants including HIV, CMV, and hepatitis.

Additionally, an advantage of the method of the present invention isthat it provides a sample of leukocytes that has a low level of redblood cell contamination that allows the sample to be used directly inPCR analysis. Further, by using tubes to store the leukocytes, the tubescan be labeled with, for example, bar codes that will decrease thepossibility of mislabeling samples.

By way of example, and not limitation, an example of the presentinvention will now be set forth.

EXAMPLE NO. 1

This experiment validates the methodology that isolates leukocytes usingthe device and method illustrated in FIGS. 3-5 from a buffy coatpreparation obtained using an Optipress® apparatus and Optipak®disposables, hereinafter, for the sake of brevity, referred to as theOptipress® device. The studies focussed on the collection of a leukocyterich sample from a mixed population of blood cells (erythrocytes,platelets, leukocytes) using the syringe chamber of FIG. 3 at differentcentrifugation forces and times.

Cell Sources

Buffy coats were prepared from whole blood units of various agespurchased from Interstate Blood Bank or from in-house donors using theOptipress® device. Briefly, the whole blood units were centrifugedat >3500×g (High Speed) or processed using the Optipress system. Thehigh speed buffy coat preparation (35-50 mls) contains leukocytes andhigh concentrations of platelets and red blood cells (mean HCT=⁻ 55%).The slow speed buffy coat preparations contain leukocytes with lowerlevels of contaminating platelets and red blood cells (mean HCT=42.4%).The buffy coats were held at room temperature until used in theseparation protocols.

Monoclonal Antibodies

Mouse IgG monoclonal antibodies conjugated with fluorescein (FITC) orphycoerythrin (PE) were purchased from Becton Dickinson, Mountain View,Calif. The monoclonal antibodies with specificity to: CD3 (Leu 4), CD4(Leu 3), CD8 (Leu 2a), CD14 (Leu M3), CD16 (Leu 11c), CD11b (anti-Cr3),CD19 (Leu12) or CD45 (HLe) were used to measure the distribution of thedifferent leukocyte subpopulations before and after separation in thesyringe chamber.

FACS Lysing Solution

FACS lysing solution was purchased from Becton Dickinson, Mountain View,Calif. as a 10× concentrate and diluted to a working 1× solution withSterile Water prior to use. The lysing reagent is used to lyse red bloodcells following direct immunofluorescence staining of whole blood orblood products (blood products with high levels of RBC contamination)with monoclonal antibodies prior to flow cytometric analysis.

Modified Syringe Leukocyte Collection Chamber

The modified syringe leukocyte collection chamber uses a 5 ml disposableplastic syringe illustrated in FIG. 3. Other components of the deviceare a detachable syringe sleeve, tubing, and a sample collection tube.Leukocyte separation within the chamber is based on the differentdensities of the cellular components within blood.

Cell Separation Methods

Different centrifugation forces and times were evaluated when performingthe leukocyte separations using either high or slow speed buffy coatpreparations.

The chamber was loaded by drawing a 4 ml sample of the buffy coatfraction obtained using the Optipress® device into the chamber. Thesyringe sleeve was then attached to the plunger to prevent its collapseduring centrifugation, and the chamber was placed into a 50 mlcentrifuge tube. Slow speed buffy coat preparations were centrifuged for5, 7, and/or 10 minutes at speeds ranging between 500-1000×g. High speedbuffy coat preparations were separated within the syringe chambers atcentrifugation speeds between 500-1500×g for 5, 7, and/or 10 minutes.

Following centrifugation, a piece of tubing was attached to the samplecollection port and the plasma, platelets and buffy coat layer wereexpressed into a sample collection tube. A hemostat was clamped onto thetubing when the packed RBC layer were observed entering the tubing. Thesample tubing was then removed from the chamber and allowed to draininto a sample tube. The leukocyte enriched samples were held at roomtemperature until counted and stained for FACS analysis.

Phenotypic Analysis of Cells

Cells from the pre and post modified syringe leukocyte collectionchamber samples were transferred into appropriately labeled 12×75 mmpolypropylene tubes and stained with the following monoclonal antibodycombinations: HLeFITC, CD3PE/HLeFITC, CD4PE/HLeFITC, CD8PE/HLeFITC,CD14PE/HLeFITC, CD16PE/HLeFITC, Cr3PE/HLeFITC and CD19PE/HLeFITC. Thecells were incubated for 15-20 minutes at room temperature following theaddition of the monoclonal antibody.

After the staining period, the cells were resuspended in 1-2 mls of 1×FACS lysing solution and incubated 10 minutes at room temperature. Cellswere collected by centrifugation 2 minutes high speed using a Serofugecentrifuge. The supernates were poured off and the cells washed 2× withPAB. After the last wash, the cells were resuspended in PAB and held onice or refrigerated overnight until analyzed on the FACSCAN flowcytometer.

Results

The results are divided into two sections. Section I summarizes theleukocyte isolation from slow speed buffy coat preparations and SectionII summarizes the leukocyte isolation from high speed buffy coatpreparations.

I. Leukocyte Isolation Using Modified Syringe Leukocyte CollectionChamber of FIG. 3

A. Isolation of leukocytes from slow speed buffy coat preparationsprepared from 24 hour old blood received from Interstate Blood Bank,using the modified syringe leukocyte collection chamber of the presentinvention.

A total of eight slow speed buffy coat preparations obtained using theOptipress® device were processed using the syringe chamber of FIG. 3.Table 1 summarizes the WBC, RBC, and platelet cell counts pre and postprocessing of the buffy coats within the chamber at differentcentrifugation forces and times.

The mean WBC concentration of the raw buffy coat samples was 3.99×10⁷WBC/ml. Processing the buffy coats at 1000×g for 5 minutes gave thehighest mean number of WBC/ml recovered (5.54×10⁶). Centrifugation for 7minutes at 1000×g gave the poorest WBC yield (Mean=1.8×10⁶), while the10 minute sample gave an intermediate amount of WBC's (Mean=3.4×10⁶).The red blood cell (RBC) concentration decreased significantly with theincreasing centrifugation times. A decrease in the RBC concentration of1.8 logs was observed in the 5 minute, 2.2 logs in the 7 minute and 5.2logs in the 10 minute 1000×g post chamber samples as compared to the prechamber sample. The platelet concentrations in the post chamber sampleswere slightly greater than the pre chamber value.

Decreasing the centrifugation force to 750×g resulted in mean WBC/mlconcentrations of 1.225×10⁶, 1.7×10⁶, and 3.825×10⁶ using 5, 7, and 10minute centrifugation respectively. The RBC contamination in the postchamber samples collected at 750×g decreased with increasingcentrifugation times (5 minutes=1.86 logs; 7 minutes=2.45 logs; 10minutes=5.27 logs). Platelet concentrations in the 750×g post chambercollected samples were slightly higher than the pre chamber controlsample.

Processing the buffy coat preparations at 500×g for 5 minutes did notalways result in the formation of a distinct WBC layer within thechamber. Therefore, a 5 minute centrifugation time at 500×g is notrecommended for collecting leukocytes with the syringe chamber for PCRanalysis. Centrifugation at 500×g for 7 and 10 minutes resulted in meanWBC/ml concentrations of 3.42×10⁶ and 1.64×10⁶ respectively. Increasingthe centrifugation time from 7 to 10 minutes resulted in a significantdecrease in the RBC contamination in the post chamber samples at 500×g.RBC contamination in the 10 minute sample was reduced 5.09 logs whilethe 7 minute sample had a 1.83 low reduction in RBC concentration ascompared to the pre chamber sample. The platelet concentrations wereslightly elevated in both the 7 and 10 minute 500×g post syringe chambersamples as compared to the pre chamber value.

Table 2 summarizes the phenotypes of the cells pre and post processingof the buffy coats using the syringe chamber. To analyze the major cellpopulations (lymphocytes, monocytes and granulocytes) an electronic gatewas set to include the HLe (pan leukocyte) positive cells and excludethe HLe negative cells from the analysis. After the HLe gate was set,electronic regions were set around the lymphocyte, monocyte andgranulocyte populations based on their light scattering properties (FSCvs SSC).

The lymphocyte population in the pre chamber buffy coat samples rangedfrom 9.18%-61.93% with a mean value of 34.36%±16.62%. The monocytepopulation had a mean value of 7.04%±7.42% (range=0.19%-22.07%) whilethe granulocyte population had a mean value of 54.47%±19.05% (range:20.85%-76.28%). Samples collected from processing of the buffy coatswith the syringe chamber at 1000×g (see FIG. 6) resulted in an increasein the lymphocyte population and a decrease in the granulocytepopulation. However, increasing the time of centrifugation from 5 to 7to 10 minutes decreased the mean lymphocyte percentage (5 minutes=79.59:7 minutes=71.28%: 10 minutes=70.60%) and increased the mean granulocytepercentage (5 minutes=12.42%: 7 minutes=13.66%: 10 minutes=17.62% in thepost syringe chamber samples. The monocyte percentage decreased slightlyin the 5 minute sample (mean=3.18%) and remained similar to the prechamber percentage (7.04%) at 7 (7.26%) or 10 (6.53%) minutes.

FIG. 7 graphically illustrates samples collected from the syringechamber at 750×g for 5, 7, and/or 10 minutes. The lymphocyte percentagefor each of the centrifugation times tested increased while thegranulocyte percentage decreased as compared to the pre buffy coatcontrol samples. Samples collected from the chambers after 5 or 7minutes of centrifugation had similar lymphocyte (5 minutes=80.93%: 7minutes=79.01%), monocyte (5 minutes=2.20%: 7 minutes=2.67%), andgranulocyte (5 minutes=11.71%: 7 minutes=13.02%) percentages. Samplescollected at 750×g for 10 minutes had lower lymphocyte (67.38%) andhigher monocyte (4.29%) and granulocyte (20.50%) percentages.

Post syringe chamber samples collected at 500×g for 7 or 10 minutesdisplayed increased lymphocyte and decreased granulocyte percentages(see FIG. 8). Samples collected from the chamber at 500×g for 7 minuteshad an increased lymphocyte percentage (75.45%), and decreasedpercentages of monocytes (2.31%) and granulocytes (15.69%) as comparedto the control buffy coat samples. Increasing the centrifugation time to10 minutes at 500×g resulted in a lower percentage of lymphocytes(57.77%) and increased percentages of monocytes (8.40%) and granulocytes(27.32%) as compared to the 7 minute samples.

Conclusions

1) All samples collected from the modified syringe leukocyte collectionchamber of FIG. 3 had increased lymphocyte percentages regardless of thecentrifugation force or time as compared to the pre chamber buffy coatcontrols.

2) With any of the centrifugation forces tested (500, 750, 1000×g) anincrease in the centrifugation time resulted in a decrease in thepercentage of granulocytes in the post chamber samples. The percentageof monocytes, however, remained fairly constant regardless of thecentrifugational force or time.

3) A centrifugational force of at least 500×g for 7 minutes is requiredto form a distinct buffy coat layer within the syringe chamber for PCRsample collection.

4) Increasing the time of centrifugation at any of the gravitationalforces tested resulted in a decrease in the RBC contamination of the PCRsample collected.

5) Decreasing the centrifugational force and/or time increases thenumber of platelets in the PCR sample collected from the syringechamber.

6) It appears that a centrifugation force of 750×g for 7 minutes givesthe best sample for PCR diagnostics (highest lymphocyte percentage, lowRBC contamination) when using the modified syringe leukocyte collectionchamber of FIG. 3.

The modified syringe leukocyte collection chamber of FIG. 3 allows thecollection of leukocytes from a buffy coat preparation obtained at aslow speed that is enriched for cells displaying lymphocyte lightscattering characteristics. A centrifugation force of 750×g for 7minutes appears to be the optimal conditions required to generate asample for PCR testing. This sample for use in PCR diagnostics isenriched to a mean lymphocyte percentage of >80% and also has reducedRBC contamination.

B. Isolation of leukocytes from buffy coat preparations prepared fromfresh <4 hour old blood received from in-house Fenwal donors obtainedusing an Optipress® device, using the modified syringe leukocytecollection chamber of FIG. 3.

Two experiments were performed to determine if the storage buffy coatfractions prior to processing with the modified syringe leukocytecollection chamber, would effect the chamber's performance. In the firstexperiment, a buffy coat fraction was received that had been processedusing an Optipress® device from fresh in-house blood (<4 hours old). Thebuffy coat fraction was processed in syringe chambers at 500, 400, 300,200, and 100×g for 10 minutes. Table 3a summarizes the cell counts preand post processing of the buffy coat with the syringe chamber.

The white blood cell (WBC) concentration of the samples collected fromthe chambers increased with the decrease in the centrifugation forceused to process the cells. The RBC concentration was the lowest in the400×g sample (7×10⁷) and the highest in the 100×g (2×10⁸) sample.

FIG. 9a illustrates the phenotyping results of the samples pre and postprocessing with the syringe chambers. The lymphocyte concentrationincreased from the pre chamber buffy coat control percentage of 31.08%to 80.53% at 500×g, 83.28% at 400×g, 88.70% at 300×g, 74.59% at 200×g,and 57.75% at 100×g. Corresponding to the increased lymphocyteconcentration a decrease in the percentage of granulocytes in each postsyringe chamber samples was observed. The monocyte concentrationfluctuated from a pre chamber control value of 8.56% to post chamberconcentrations of 3.95% at 300×g, 5.25% at 400×g, 8.47% at 200×g, 10.53%at 500×g, and 12.06% in the 100×g sample. The unused portion of thebuffy coat was allowed to sit at room temperature for 48 hours beforebeing processed at 400×g for 10 minutes using a syringe chamber. WBC,RBC and Platelet concentrations in the 48 hour old buffy coat samplewere comparable to the fresh buffy coat sample concentrations (Table3a). The 48 hour old buffy coat was processed in duplicate at 400×g for10 minutes to determine if there was any variability between the samplescollected from the chamber.

Table 3b sets forth the cell counts of the samples pre and postprocessing with the chambers. The WBC, RBC and Platelet concentrationsof the 400×g #1 and the 400×g #2 samples were similar. However, whencompared to the fresh sample collected at 400×g, there was a 5 foldincrease in the number of WBC/ml collected with the 48 hour old sample.The mean RBC contamination was 2.5 fold higher in the 48 hour ascompared to the fresh sample. The mean platelet concentration (5.06×10⁸)was slightly lower than the platelet concentration in the freshlyprocessed sample (5.49×10⁸).

FIG. 9b illustrates the phenotyping data of the 48 hour old buffy coatsample pre and post processing with the syringe chamber of FIG. 3. Therewere no apparent differences in the percentages of lymphocytes,monocytes, or granulocytes between the 48 hour old buffy coat controlsample and the 400×g 10 minutes syringe chamber samples.

Comparing the freshly processed buffy coat and the 48 hour old buffycoat, the lymphocyte and monocyte percentages decreased while thegranulocyte percentage increased over the storage time of the buffycoat.

In the second experiment, a buffy coat fraction was received that hadbeen processed from fresh in-house blood (<4 hours old) using anOptipress® device. The buffy coat fraction was processed in duplicateusing syringe chambers at 500, 400, 300, and 200×g for 10 minutes.

Table 4 summarizes the cell counts pre and post processing of the buffycoat with the syringe chamber. The mean, WBC concentration was thehighest with the 200×g sample (1.66×10⁷) followed by the 400×g(1.15%×10⁷), 300×g (9.1%×10⁶), and the 500×g (5.4×10⁶) post chambersamples. The RBC contamination in the post chamber samples were reducedfrom a pre chamber value of 4.76%×10⁹ to 6.5×10⁷ at 500×g, 9.0×10⁷ at400×g, 8.5×10⁷ at 300×g and 9.0×10⁷ at 200×g. Platelet concentrationswere slightly higher in all of the post chamber samples as compared tothe pre chamber value of 2.75×10⁸ platelets/ml.

FIG. 10a illustrates the phenotyping results of the samples pre and postprocessing with the syringe chambers. The lymphocyte concentrationincreased from a pre chamber buffy coat control percentage of 42.05%to >82% in all post chamber samples. The monocyte concentration in allthe post chamber samples were comparable to the 8.09% in the pre chambersample. The granulocyte population decreased from a pre chamberconcentration of 48.93% to <8.0% in the post chamber samples. Samplescollected at the different centrifugation forces showed littlevariability between the duplicate samples for both the cell counts(WBC/ml, RBC/ml, Platelet/ml) and phenotype data (% Lymphocytes, %Monocytes, % Granulocytes). The buffy coat fraction was allowed to sitovernight (⁻ 24 hours) at room temperature before reprocessing with thesyringe chamber at 400×g for 10 minutes. Cell counts were not performedon the 24 hour old buffy coat or the post 400×g syringe chamber sample.

FIG. 10b illustrates the phenotype of the samples pre and postprocessing of the 24 hour old buffy coat with the syringe chamber. Thepercentage of lymphocytes increased from 44.07% in the 24 hour prechamber buffy coat control sample to 60.74% in the 400×g syringe chambersample. The Granulocyte percentage decreased from 46.20% to 27.68% inthe post chamber sample. The monocyte concentration remained relativelyunchanged between the pre chamber (7.77%) and the post chamber sample(9.57%).

Storage of the buffy coat sample, collected using the Optipress® device,for 24 hours at room temperature did not change the phenotype of thecells in the buffy coat fraction (Fresh BC: Lymphocytes=42.05%; 24 hourBC: Lymphocytes=44.07%; Fresh BC: Monocytes=8.09%; 24 hour BC:Monocytes=7.77%; Fresh BC: Granulocytes=48.93%; 24 hour BC:Granulocytes=46.20%). However, storage of the buffy coat overnight for24 hours did have an effect on the percentages of lymphocytes harvestedwith the chamber at 400×g for 10 minutes. The lymphocyte percentage inthe post chamber sample was decreased from a mean value of 87.7% in thefresh sample to 60.74% in the 24 hour old buffy coat. The granulocytepopulation increased from a mean percentage of 1.59% in the fresh sampleto 27.68% in the 24 hour buffy coat post processing with the syringechamber. The monocyte percentage in the post chamber fractions wereunaffected by the storage of the buffy coat for 24 hours (Fresh=9.23%:24 hour=9.57%).

Conclusions

1) The use of "fresh" buffy coat fractions prepared from <4 hour oldwhole blood resulted in the collection of samples from the modifiedsyringe leukocyte collection of FIG. 3 with >80% lymphocytes.

2) Centrifugation of the syringe chambers at 300-500×g for 10 minutesappears to yield the best samples for PCR diagnostics. Sufficient WBCnumbers (>1×10⁶), low RBC contamination, and high lymphocyte numbers(>80%) make these samples acceptable for PCR testing.

3) RBC concentration (<1×10⁸ /ml) was greatly reduced followingprocessing of the "fresh" buffy coats in the syringe chambers.

4) Platelet concentrations are slightly higher in the post syringechamber samples as compared to the unprocessed pre chamber buffy coatsample. Platelet concentrations collected within the chamber should notaffect the PCR reaction.

5) Storage of the buffy coat samples for 24 to 48 hours did not affectthe cell counts (WBC/ml, RBC/ml, Platelet/ml) or the phenotypes (%Lymphocytes, % Monocytes, % Granulocytes) as compared to the Day 0"fresh" buffy coat samples. However, the storage of the buffy coats for24 or 48 hours did decrease the percentage of lymphocytes in the postsyringe chamber samples collected at 400×g for 10 minutes. Thelymphocyte percentage decreased from 83.28% in the fresh syringe chambersample to 41.42% in the 48 hour sample. After 24 hours the lymphocytepercentage decreased to 60.74% from a Day 0 lymphocyte percentage of87.7%.

II. Leukocyte Isolation Using Modified Syringe Leukocyte CollectionChamber of FIG. 3

Isolation of leukocytes from high speed buffy coat preparations preparedfrom 24 hour old blood received from Interstate Blood Bank, using themodified syringe leukocyte collection chamber of FIG. 3.

A total of four high speed (>3500×g) Optipress® device buffy coatpreparations were processed using the modified syringe leukocytecollection chambers of FIG. 3. Cell counts were performed using a SysmexK-1000 automated cell counter on both the pre chamber buffy coats andthe post syringe chamber collected samples. Detailed phenotypic analysiswas also performed on the pre and post chamber samples to characterizethe lymphocyte subpopulations (CD3, CD4, CD8, NK, CD19) along themonocyte and granulocyte populations.

Table 5 summarizes the cell counts of the raw buffy coat pre chambersamples and the post syringe chamber samples collected at differentcentrifugation forces and times. The mean WBC concentration of the prechamber samples was 2.615×10⁷ /ml. Post chamber collected samplesdisplayed a decrease in WBC concentration with a decrease in bothcentrifugation force and time. All the post chamber collected samplescontained greater than 1×10⁶ WBC's used in a typical PCR digestionreaction. Decreasing the time and force of the centrifugation resultedin increased levels of RBC contamination in the post chamber samples.The platelet concentration in the post chamber collected samples wasoften above the detectable limits of the Sysmex K-1000 cell counter.However, the total number of RBC's and/or platelets that would bepresent in a 1×10⁶ WBC sample would not interfere with the PCR reaction.A centrifugation time of either 5 or 7 minutes at 750×g appears to yieldthe best sample for PCR analysis. These samples gave sufficient WBCnumbers (>1×10⁶) with acceptable levels of RBC's (≦5×10⁸) and/orplatelets (≦5×10⁹) to be used directly for PCR analysis.

Table 6 summarizes the phenotypes of the post chamber samples. Only asingle sample was processed in the chamber at 1500×g for 7 or 10minutes. In the 1500×g 7 minute post chamber sample the percentage ofCD3 (T-lymphocytes), CD4 (T-helper), CD8 (T-suppressor) and CD14(monocytes) cells increased slightly as compared to the pre chamberpercentages. Natural Killer cells (NK, CD16) and B-lymphocytes (CD19)increased from pre chamber percentages of 6.34% and 5.66% to postchamber values of 11.66% and 10.79% respectively. Granulocytes (CD16)decreased from 35.29% to 6.92% after processing of the high speed buffycoat fraction with the syringe chamber at 1500×g for 7 minutes.Increasing the centrifugation time to 10 minutes at 1500×g resulted inpost chamber samples having percentages of CD3, CD4, CD8, CD16, CD19,CD14 and CD16 cells similar to the pre chamber buffy coat values.

Optipress® device buffy coat samples were processed in the modifiedsyringe leukocyte collection chambers of FIG. 3 at 1000×g for 5, 7, and10 minutes. The CD3+ T-lymphocyte percentages were increased in the 5minute sample (mean=41.67%), similar at 7 minutes (mean=34.24) anddecreased in the 10 minute samples (17.87%) as compared to the prechamber samples (mean=34.47%). T-helper (CD4) and T-suppressor (CD8)values were similar to their pre chamber values. CD16+ NK cellsincreased from a pre chamber value of 6.34% to 12.07%, 12.47%, and17.35% in the 5, 7, and 10 minutes post chamber samples respectively.Samples processed for 5 minutes at 1000×g had a Cr3+ percentage(mean=12.40%) similar to the pre chamber samples percentage (mean12.79%). Post chamber Cr3+ (NK percentages were increased in the 7minute (mean=23.19%) and 10 minute (mean=20.30%) samples. B-lymphocytesincreased from 6.23% to 14.26% in the 5 minute, 14.57% in the 7 minute,to 24.85% in the 10 minute post chamber samples (%=mean values).Monocyte percentages were slightly elevated in the 5 and 10 minutesamples (pre mean=10.96%: 5 minutes mean=13.17; 10 minutes mean=13.06%)and 1.9× greater in the 7 minute (mean=20.85%) post chamber sample. Theamount of granulocytes (pre chamber mean=38.57%) decreased to 11.30%(mean) in the 5 minute, 25.86% (mean) in the 7 minute, and 19.27% (mean)in the 10 minute post chamber samples centrifuged at 1000×g.

Samples processed at 750×g for 5 minutes had a decrease in theT-lymphocyte number from a pre chamber mean of 34.47% to a post chambervalue of 26.85%. T-lymphocyte values in the 7 or 10 minutes post chambersamples gave CD3+ values similar to the pre chamber percentage (7minutes mean=31.69%; 10 minutes mean=30.05%). An increase in the CD4+,T-helper cells, percentages was not observed in any of threecentrifugation times tested. The CD8+, T-suppressor cells, also did notshow an enrichment in any of the post chamber samples collected at 750×gfor 5, 7, or 10 minutes. NK cells (Cr3+) increased in each of the postchamber samples. Cr3+ NK cells increased 1.4× the pre chamber in the 5minute samples (pre mean=12.79%; 5 minute mean=18.01%). Also, the Cr3+percentage in the 7 (mean=25.75%) and 10 (mean=25.40%) minute samplesincreased >2× the pre chamber mean Cr3+ percentage (mean=12.79%). Anenrichment in the B-lymphocyte, CD19+, percentage was observed in all ofthe post chamber samples collected at 750×g. Monocyte, CD14+,percentages were also greater than the pre chamber percentage for eachof the different centrifugation times. The 5 minute post chamber sampleshad the highest CD14+ percentage 26.76%. Increasing the centrifugationtime resulted in a decrease in the CD14+ percent (7 minute mean=20.3.1%;10 minute mean=14.66%) but were still greater than the pre chambersample CD14+ percentage. A decrease in granulocyte contamination wasobserved for all the post chamber samples. The Cr3+ granulocyte percentdecreased from a pre chamber value of 38.57% (mean) to 5.16% (mean) inthe 5 minute, 7.48% (mean) 7 minute, and 16.50% (mean) in the 10 minutepost chamber samples.

Optipress® device buffy coat fractions were also processed in themodified syringe leukocyte collection chamber of FIG. 3 at 500×g for 7or 10 minutes. No conclusions could be made in the 7 minute sample dueincomplete phenotyping results. The CD4+ percentage in this sample(23.35%) was similar to the pre chamber percentage (7-7:I 26.61%). TheT-suppressor value was also similar to pre chamber value (Pre=17.33; 7minute=20.84%). The CD14+ monocyte percentage did increase from a prechamber value of 7.37% to a post chamber value of 15.05%. No increase inthe percentage of CD3+, CD4+, CD8+, CD16+ or Cr3+ (NK) cells wereobserved in the post syringe chamber samples collected at 500×g for 10minutes. The B-lymphocyte percentage increased from 6.53% (mean) to11.51%, while the monocyte percentage increased from 10.96% (mean) to24.95% (mean). A decrease in the number of Cr3+ granulocytes wasobserved (pre mean=38.57%; 10 minute mean=12.18%) in the 10 minute postchamber samples.

Conclusions

1) High speed Optipress® device buffy coat fractions processed using themodified syringe leukocyte collection chamber of FIG. 3 had plateletconcentrations (≦5×10⁹) and/or RBC concentrations (≦5×10⁸) that wouldnot inhibit PCR reactions (1×10⁶ WBC/PCR digestion reaction).

2) The total number of WBC's collected from the syringe chambers weresufficient to perform a typical PCR digestion reaction (1×10⁶ WBC).

3) T-lymphocyte (CD3+) concentrations were not enriched for in the postchamber samples except in the 1000×g 5 minute samples where an increasedpercentage of CD3+ cells was observed. The CD3+ percentage in the otherpost syringe chamber collected samples were similar to the CD3+percentage in the pre chamber high speed optipress buffy coat fractions.

4) The CD4+ T-helper cell subpopulation was not enriched in the postchamber collected samples at any of the centrifugation forces or timestested. CD8+ T-suppressor cells also were not enriched in the final postchamber samples.

5) Natural Killer cell (Cr3+) percentages were increased in the 7 and 10minute 1000×g and also all of the 750×g post chamber collected samples.

6) An enrichment in the concentration of B-lymphocytes (CD19+) wasobserved in all the post chamber collected samples. Decreasing thecentrifugation force from 1000×g to 750×g increased the CD19+ percentagein the post chamber samples.

7) Monocytes (CD14+) showed slight increases in their percentages in the5 and 10 minute 1000×g collected samples. The 1000×g 7 minute sampleshad a 1.9 fold increase in CD14+ cells over the pre chamber high speedOptipress buffy coat samples. Post chamber samples collected at 750×galso displayed increased CD14+ concentrations as compared to the prechamber fractions. However, a decrease in the CD14+ percentage wasobserved with an increase in the centrifugation time at 750×g.

8) Granulocyte contamination (Cr3+) was decreased in all the postchamber collected samples.

9) The optimal centrifugation force required to process a high speedOptipress® device buffy coat fraction in the syringe chamber for PCRtesting appears to be 750×g. A centrifugation time of 5-7 minutesappears to be required to collect the best PCR sample.

SUMMARY

The modified syringe leukocyte collection chamber of FIG. 3 offers aconvenient method to collect a leukocyte rich sample from a mixedpopulation of blood cells for use in PCR screening of blood for viralcontamination. The optimal speed required to collect a PCR sample fromeither a slow speed (<3000 rpm) or a high speed (>3500 rpm) Optipress®device buffy coat preparation was 750×g. These samples would providesufficient numbers of WBC's (1×10⁶) with minimal RBC (≦5×10⁸) and/orplatelet (≦5×10⁹) contamination for use directly in the PCR analysis.

However, slow speed Optipress® device buffy coat fractions had a greaterpercentage of lymphocytes in the post syringe samples (750×g>45%). Thisincreased lymphocyte concentration may increase the sensitivity of PCRanalysis for HIV infection. The use of slow speed buffy coat fractionsprepared from fresh blood (<4 hours old) may also increase WBC recoveryand lymphocyte purity in the post syringe chamber samples. Storage ofthe buffy coat for 24-48 hours decreases the efficiency of the chamberto collect lymphocyte enriched samples.

In summary, the modified syringe leukocyte collection chamber of FIG. 3offers a quick and convenient method of collecting leukocyte rich PCRsamples using Optipress® device buffy coat preparations.

                                      TABLE 1                                     __________________________________________________________________________    Summary of cell courts pre and post processing of slow speed Optipress        ®                                                                         device buffy coat preparations with the Modified Syringe Leukocyte            Collection                                                                    Chamber.                                                                      __________________________________________________________________________    Raw Buffy Coat: Pre Chamber Values                                                                               mis                                        Sample Id                                                                          Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-15:A                                                                             --   28,400,000                                                                           4,300,000,000                                                                        277,000,000                                                                          35.9                                                                              --                                         5-15:C                                                                             --   66,400,000                                                                           4,450,000,000                                                                        602,000,000                                                                          58.6                                                                              --                                         5-19:A                                                                             --   22,200,000                                                                           5,420,000,000                                                                        230,000,000                                                                          42.9                                                                              --                                         5-28:                                                                              --   39,000,000                                                                           3,790,000,000                                                                        387,000,000                                                                          38.1                                                                              --                                         6-2:A                                                                              --   27,700,000                                                                           4,490,000,000                                                                        188,000,000                                                                          42.9                                                                              --                                         6-2:B                                                                              --   34,000,000                                                                           4,060,000,000                                                                        215,000,000                                                                          40.4                                                                              --                                         6-2:D                                                                              --   63,800,000                                                                           3,630,000,000                                                                        577,000,000                                                                          39.3                                                                              --                                         6-2:E                                                                              --   37,700,000                                                                           4,900,000,000                                                                        287,000,000                                                                          41.3                                                                              --                                              mean 3.990E + 07                                                                          4.380E + 09                                                                          3.454E + 08                                                                          42.4                                                                              --                                              Std  1.544E + 07                                                                          5.464E + 08                                                                          1.518E + 08                                                                          6.5 --                                         __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 1000 xg                   Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-28:                                                                              5 Minutes                                                                          6,400,000                                                                            70,000,000                                                                           540,000,000                                                                          0.4 1.45                                       6-2:A                                                                              5 Minutes                                                                          9,000,000                                                                            100,000,000                                                                          301,000,000                                                                          0.7 2.10                                       6-2:B                                                                              5 Minutes                                                                          9,600,000                                                                            90,000,000                                                                           346,000,000                                                                          0.6 2.20                                       6-2:D                                                                              5 Minutes                                                                          2,300,000                                                                            100,000,000                                                                          932,000,000                                                                          0.5 2.10                                       6-2:E                                                                              5 Minutes                                                                          400,000                                                                              60,000,000                                                                           547,000,000                                                                          0.3 2.60                                            Mean 5.540E + 06                                                                          8.400E + 07                                                                          5.332E + 08                                                                          0.5 2.09                                            Std  3.636E + 06                                                                          1.625E + 07                                                                          2.228E + 08                                                                          0.1 0.37                                       __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 100 xg                    Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-28:                                                                              7 Minutes                                                                          4,200,000                                                                            60,000,000                                                                           540,000,000                                                                          0.4 1.45                                       6-2:A                                                                              7 Minutes                                                                          2,900,000                                                                            5,000,000                                                                            301,000,000                                                                          0.7 2.10                                       6-2:B                                                                              7 Minutes                                                                          1,500,000                                                                            30,000,000                                                                           346,000,000                                                                          0.6 2.20                                       6-2:D                                                                              7 Minutes                                                                          200,000                                                                              50,000,000                                                                           932,000,000                                                                          0.5 2.10                                       6-2:E                                                                              7 Minutes                                                                          200,000                                                                              30,000,000                                                                           547,000,000                                                                          0.3 2.60                                            Mean 1.800E + 06                                                                          3.500E + 07                                                                          4.204E + 08                                                                          0.2 2.01                                            Std  1.561E + 06                                                                          1.897E + 07                                                                          1.882E + 08                                                                          0.1 0.22                                       __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 1000 xg                   Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-28:                                                                              10 Min-                                                                            8,500,000                                                                            60,000 491,000,000                                                                          0.4 1.80                                            utes                                                                     6-2:A                                                                              10 Min-                                                                            2,800,000                                                                            30,000 95,000,000                                                                           0.2 1.70                                            utes                                                                     6-2:B                                                                              10 Min-                                                                            3,000,000                                                                            20,000 202,000,000                                                                          0.1 1.90                                            utes                                                                     6-2:D                                                                              10 Min-                                                                            200,000                                                                              30,000 699,000,000                                                                          0.1 1.90                                            utes                                                                     6-2:E                                                                              10 Min-                                                                            1,000,000                                                                            30,000 307,000,000                                                                          0.1 2.10                                            utes                                                                          Mean 2.100E + 06                                                                          3.400E + 04                                                                          3.588E + 08                                                                          0.2 1.88                                            Std  2.901E + 06                                                                          1.356E + 04                                                                          2.145E + 08                                                                          0.1 0.13                                       __________________________________________________________________________    Raw Buffy Coat: Pre Chamber Values                                                                               mis                                        Sample ID                                                                          Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-15:A                                                                             --   28,400,000                                                                           4,300,000,000                                                                        277,000,000                                                                          35.9                                                                              --                                         5-15:C                                                                             --   66,400,000                                                                           4,450,000,000                                                                        602,000,000                                                                          58.6                                                                              --                                         5-19:A                                                                             --   22,200,000                                                                           5,420,000,000                                                                        230,000,000                                                                          42.9                                                                              --                                         5-28:                                                                              --   39,000,000                                                                           3,790,000,000                                                                        387,000,000                                                                          38.1                                                                              --                                         6-2:A                                                                              --   27,700,000                                                                           4,490,000,000                                                                        188,000,000                                                                          42.9                                                                              --                                         6-2:B                                                                              --   34,000,000                                                                           4,060,000,000                                                                        215,000,000                                                                          40.4                                                                              --                                         6-2:D                                                                              --   63,800,000                                                                           3,630,000,000                                                                        577,000,000                                                                          39.3                                                                              --                                         6-2:E                                                                              --   37,700,000                                                                           4,900,000,000                                                                        287,000,000                                                                          41.3                                                                              --                                              mean 3.990E + 07                                                                          4.380E + 09                                                                          3.454E + 08                                                                          42.4                                                                              --                                              Std  1.544E + 07                                                                          5.464E + 08                                                                          1.518E + 08                                                                          6.5 --                                         __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 750 xg                    Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-28:                                                                              5 Minutes                                                                          5,900,000                                                                            70,000,000                                                                           653,000,000                                                                          0.4 1.10                                       6-2:A                                                                              5 Minutes                                                                          100,000                                                                              30,000,000                                                                           392,000,000                                                                          0.1 2.30                                       6-2:B                                                                              5 Minutes                                                                          3,100,000                                                                            70,000,000                                                                           297,000,000                                                                          0.3 2.20                                       6-2:D                                                                              5 Minutes                                                                          1,200,000                                                                            100,000,000                                                                          989,000,000                                                                          0.3 2.30                                       6-2:E                                                                              5 Minutes                                                                          500,000                                                                              50,000,000                                                                           556,000,000                                                                          0.2 2.80                                            Mean 2.160E + 06                                                                          6.400E + 07                                                                          5.774E + 08                                                                          0.3 2.14                                            Std  2.135E + 06                                                                          2.332E + 07                                                                          2.402E + 08                                                                          0.1 0.56                                       5-28:                                                                              7 Minutes                                                                          5,500,000                                                                            60,000,000                                                                           586,000,000                                                                          0.3 1.40                                       6-2:A                                                                              7 Minutes                                                                          1,000,000                                                                            30,000,000                                                                           248,000,000                                                                          0.1 1.90                                       6-2:B                                                                              7 Minutes                                                                          500,000                                                                              30,000,000                                                                           330,000,000                                                                          0.1 2.40                                       6-2:D                                                                              7 Minutes                                                                          1,200,000                                                                            700,000                                                                              941,000,000                                                                          0.2 2.10                                       6-2:E                                                                              7 Minutes                                                                          300,000                                                                              50,000,000                                                                           531,000,000                                                                          0.2 2.50                                            Mean 1.700E + 06                                                                          3.414E + 04                                                                          5.272E + 08                                                                          0.2 2.06                                            Std  1.928E + 06                                                                          2.036E + 04                                                                          2.476E + 08                                                                          0.1 0.39                                       5-28:                                                                              10 Min-                                                                            11,000,000                                                                           30,000 447,000,000                                                                          0.2 1.75                                            utes                                                                     6-2:A                                                                              10 Min-                                                                            700,000                                                                              20,000 168,000,000                                                                          0.1 1.80                                            utes                                                                     6-2:B                                                                              10 Min-                                                                            100,000                                                                              10,000 243,000,000                                                                          0.0 2.10                                            utes                                                                     6-2:D                                                                              10 Min-                                                                            3,500,000                                                                            50,000 807,000,000                                                                          0.2 1.90                                            utes                                                                          Mean 3.825E + 06                                                                          2.750E + 04                                                                          4.163E + 08                                                                          0.1 1.89                                            Std  4.337E + 06                                                                          1.479E + 04                                                                          2.476E + 08                                                                          0.1 0.13                                       __________________________________________________________________________    Raw Buffy Coat: Pre Chamber Values                                                                               mis                                        Sample Id                                                                          Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-15:A                                                                             --   28,400,000                                                                           4,300,000,000                                                                        277,000,000                                                                          35.9                                                                              --                                         5-15:C                                                                             --   66,400,000                                                                           4,450,000,000                                                                        602,000,000                                                                          58.6                                                                              --                                         5-19:A                                                                             --   22,200,000                                                                           5,420,000,000                                                                        230,000,000                                                                          42.9                                                                              --                                         5-28:                                                                              --   39,000,000                                                                           3,790,000,000                                                                        387,000,000                                                                          38.1                                                                              --                                         6-2:A                                                                              --   27,700,000                                                                           4,490,000,000                                                                        188,000,000                                                                          42.9                                                                              --                                         6-2:B                                                                              --   34,000,000                                                                           4,060,000,000                                                                        215,000,000                                                                          40.4                                                                              --                                         6-2:D                                                                              --   63,800,000                                                                           3,630,000,000                                                                        577,000,000                                                                          39.3                                                                              --                                         6-2:E                                                                              --   37,700,000                                                                           4,900,000,000                                                                        287,000,000                                                                          41.3                                                                              --                                              mean 3.990E + 07                                                                          4.380E + 09                                                                          3.454E + 08                                                                          42.4                                                                              --                                              Std  1.544E + 07                                                                          5.464E + 08                                                                          1.518E + 08                                                                          6.5 --                                         __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 500 xg                    Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    5-28:                                                                              5 Minutes                                                                          10,000,000                                                                           80,000,000                                                                           625,000,000                                                                          0.5 ND                                         5-28:                                                                              7 Minutes                                                                          5,100,000                                                                            80,000,000                                                                           616,000,000                                                                          0.4 1.00                                       6-2:A                                                                              7 Minutes                                                                          1,300,000                                                                            60,000,000                                                                           310,000,000                                                                          0.2 2.10                                       6-2:B                                                                              7 Minutes                                                                          5,100,000                                                                            60,000,000                                                                           368,000,000                                                                          0.3 2.70                                       6-2:D                                                                              7 Minutes                                                                          5,400,000                                                                            130,000,000                                                                          1,055,000,000                                                                        0.5 2.40                                       6-2:E                                                                              7 Minutes                                                                          200,000                                                                              50,000,000                                                                           550,000,000                                                                          0.2 2.70                                            Mean 3.420E + 06                                                                          7.600E + 07                                                                          5.858E + 08                                                                          0.3 2.18                                            Std  2.210E + 06                                                                          2.871E + 07                                                                          2.834E + 08                                                                          0.1 0.63                                       5-15:A                                                                             10 Min-                                                                            24,800,000                                                                           300,000                                                                              331,000,000                                                                          2.5 ND                                              utes                                                                     5-15:C                                                                             10 Min-                                                                            129,000,000                                                                          340,000                                                                              1,282,000,000                                                                        2.4 ND                                              utes                                                                     5-15:A                                                                             10 Min-                                                                            200,000                                                                              30,000 257,000,000                                                                          0.2 ND                                              utes                                                                     5-28:                                                                              10 Min-                                                                            3,300,000                                                                            60,000 556,000,000                                                                          0.3 1.40                                            utes                                                                     6-2:A                                                                              10 Min-                                                                            400,000                                                                              20,000 226,000,000                                                                          0.1 2.10                                            utes                                                                     6-2:B                                                                              10 Min-                                                                            700,000                                                                              20,000 275,000,000                                                                          0.1 2.30                                            utes                                                                     6-2:D                                                                              10 Min-                                                                            3,200,000                                                                            90,000 945,000,000                                                                          0.3 2.00                                            utes                                                                     6-2:E                                                                              10 Min-                                                                            600,000                                                                              50,000 503,000,000                                                                          0.2 2.40                                            utes                                                                          Mean 2.028E + 07                                                                          1.138E + 05                                                                          5.469E + 08                                                                          0.8 2.04                                            Std  4.182E + 07                                                                          1.214E + 05                                                                          3.550E + 08                                                                          1.0 0.35                                       __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Summary of cell phenotypes pre end post processing of slow speed              Optipress ® device buffy coat preparations with the Modified              Syringe Leukocyte Collection Chamber.                                         Raw Buffy Cost: Pre Chamber Values                                            Phenotypes based on light scattering properties (FSC vs. SSC) of the          HLe positive cells.                                                           Sample Id                                                                             Time    Lymphocytes Monocytes                                                                             Granulocytes                              ______________________________________                                        5-15:A          45.63%      22.07%  20.85%                                    5-15:C          9.18%       11.82%  74.02%                                    5-19:A          14.89%      5.97%   76.28%                                    5-28:           39.65%      8.33%   47.36%                                    6-2:A           36.34%      0.67%   61.28%                                    6-2:D           32.88%      0.24%   65.38%                                    6-2:E           61.93%      0.19%   36.11%                                            Mean    34.36%      3.18%   54.47%                                            Std     16.62%      1.20%   19.05%                                    ______________________________________                                        Post Modified Syringe Leukocyte Collection Chamber: 1000 xg                   Sample Id                                                                            Time     Lymphocytes Monocytes                                                                             Granulocytes                              ______________________________________                                        5-28:  5 Minutes                                                                              91.64%      3.30%   0.47%                                     6-2:A  5 Minutes                                                                              64.90%      4.18%   26.35%                                    6-2:B  5 Minutes                                                                              85.51%      2.27%   10.26%                                    6-2:D  5 Minutes                                                                              77.95%      1.44%   17.39%                                    6-2:E  5 Minutes                                                                              77.96%      4.70%   7.65%                                            Mean     79.59%      3.18%   12.42%                                           Std      8.96%       1.20%   8.82%                                     5-28:  7 Minutes                                                                              81.82%      13.42%  0.98%                                     6-2:A  7 Minutes                                                                              64.63%      4.16%   28.23%                                    6-2:B  7 Minutes                                                                              81.55%      3.71%   11.73%                                    6-2:E  7 Minutes                                                                              57.12%      7.73%   13.71%                                           Mean     71.28%      7.25%   13.66%                                           Std      10.74%      3.89%   9.71%                                     5-28:  10 Minutes                                                                             82.25%      13.20%  1.10%                                     6-2:A  10 Minutes                                                                             66.05%      2.46%   29.30%                                    6-2:B  10 Minutes                                                                             68.99%      2.46%   25.09%                                    6-2:E  10 Minutes                                                                             65.11%      8.00%   14.99%                                           Mean     70.60%      6.53%   17.62%                                           Std      6.88%       4.47%   10.86%                                    ______________________________________                                        Post Modified Syringe Leukocyte Collection Chamber: 750 xg                    Sample Id                                                                            Time     Lymphocytes Monocytes                                                                             Granulocytes                              ______________________________________                                        5-28:  5 Minutes                                                                              94.30%      2.17%   0.42%                                     6-2:B  5 Minutes                                                                              91.14%      3.18%   3.36%                                     6-2:D  5 Minutes                                                                              55.68%      1.14%   36.92%                                    6-2:E  5 Minutes                                                                              82.58%      2.31%   6.12%                                            Mean     80.93%      2.20%   11.71%                                           Std      15.19%      0.72%   14.70%                                    5-28:  7 Minutes                                                                              93.00%      3.70%   0.35%                                     6-2:A  7 Minutes                                                                              78.43%      2.80%   15.46%                                    6-2:B  7 Minutes                                                                              78.56%      2.33%   14.63%                                    6-2:D  7 Minutes                                                                              66.45%      1.50%   27.26%                                    6-2:E  7 Minutes                                                                              78.59%      3.01%   7.40%                                            Mean     79.01%      2.67%   13.02%                                           Std      8.42%       0.73%   8.98%                                     5-28:  10 Minutes                                                                             78.05%      9.04%   1.55%                                     6-2:A  10 Minutes                                                                             69.94%      2.79%   22.75%                                    6-2:B  10 Minutes                                                                             61.68%      4.19%   23.19%                                    6-2:D  10 Minutes                                                                             59.86%      1.12%   34.50%                                           Mean     67.38%      4.29%   20.50%                                           Std      7.24%       2.95%   11.91%                                    ______________________________________                                        Post Modified Syringe Leukocyte Collection Chamber: 500 xg                    Sample Id                                                                            Time     Lymphocytes Monocytes                                                                             Granulocytes                              ______________________________________                                        5-28:  5 Minutes                                                                              92.41%      3.53%   0.17%                                     5-28:  7 Minutes                                                                              94.07%      2.50%   0.24%                                     6-2:A  7 Minutes                                                                              80.86%      3.78%   11.67%                                    6-2:B  7 Minutes                                                                              75.48%      2.68%   17.80%                                    6-2:D  7 Minutes                                                                              53.46%      0.88%   40.91%                                    6-2:E  7 Minutes                                                                              73.39%      1.73%   7.81%                                            Mean     75.45%      2.31%   15.69%                                           Std      13.14%      0.97%   13.84%                                    5-15:A 10 Minutes                                                                             33.45%      32.06%  28.08%                                    5-15:C 10 Minutes                                                                             8.47%       13.43%  71.29%                                    5-28:  10 Minutes                                                                             92.08%      3.90%   0.52%                                     6-2:A  10 Minutes                                                                             58.90%      2.39%   29.88%                                    8-2:B  10 Minutes                                                                             73.01%      3.36%   17.41%                                    6-2:D  10 Minutes                                                                             61.74%      1.01%   33.67%                                    6-2:E  10 Minutes                                                                             76.77%      2.63%   10.41%                                           Mean     57.77%      8.40%   27.32%                                           Std      26.21%      10.38%  20.99%                                    ______________________________________                                    

                  TABLE 3a                                                        ______________________________________                                        Summary of the cell counts pre and post processing of Optipress ®         device buffy coat preparation with the modified syringe leukocyte             collection chamber.                                                           Fresh Optipress ® device buffy coat prepared from <4 hour old blood.      10 Minute                                                                              Summary of Cell Counts                                               Centrifugation                                                                         w/ Sysmex K-1000                                                     Sample Id                                                                              WBC/ml    RBC/ml     Plt/ml  HCT                                     ______________________________________                                        Pre (Control)                                                                          38,300,000                                                                              4,880,000,000                                                                            318,000,000                                                                           48.5%                                   500 xg    8,800,000                                                                                100,000,000                                                                            502,000,000                                                                           0.6%                                    400 xg    6,400,000                                                                                70,000,000                                                                             549,000,000                                                                           0.3%                                    300 xg   13,400,000                                                                                90,000,000                                                                             371,000,000                                                                           0.5%                                    200 xg   23,600,000                                                                                120,000,000                                                                            547,000,000                                                                           0.8%                                    100 xg   81,000,000                                                                                200,000,000                                                                            469,000,000                                                                           1.8%                                    ______________________________________                                    

                  TABLE 3b                                                        ______________________________________                                        Summary of the cell counts pre and post processing of Optipress ®         device buffy coat preparation with the modified syringe leukocyte             collection chamber.                                                           48 hour old Optipress ® device buffy coat stored at room                  temperature.                                                                  10 Minute                                                                              Summary of Cell Counts                                               Centrifugation                                                                         w/Sysmex K-1000                                                      Sample Id                                                                              WBC/ml    RBC/ml     Plt/ml  HCT                                     ______________________________________                                        Pre (Control)                                                                          35,400,000                                                                              5,000,000,000                                                                            309,000,000                                                                           53.8%                                   400 xg #1                                                                              37,300,000                                                                                190,000,000                                                                            509,000,000                                                                           1.5%                                    400 xg #2                                                                              42,300,000                                                                                160,000,000                                                                            503,000,000                                                                           1.2%                                    ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Summary of the cell counts pre and post processing of Optipress ®         device buffy coat preparation with the modified syringe leukocyte             collection chamber.                                                           Fresh Optipress ® device buffy coat prepared from <4 hour old blood.      10 Minute                                                                              Summary of Cell Counts                                               Centrifugation                                                                         w/Sysmex K-1000                                                      Sample Id                                                                              WBC/ml    RBC/ml     Plt/ml  HCT                                     ______________________________________                                        Pre (Control)                                                                          28,100,000                                                                              4,760,000,000                                                                            275,000,000                                                                           43.9%                                   500 xg #1                                                                               4,700,000                                                                                60,000,000                                                                             381,000,000                                                                           0.3%                                    500 xg #2                                                                               6,100,000                                                                                70,000,000                                                                             447,000,000                                                                           0.4%                                    400 xg #1                                                                              14,300,000                                                                                100,000,000                                                                            539,000,000                                                                           0.5%                                    400 xg #2                                                                               8,700,000                                                                                80,000,000                                                                             526,000,000                                                                           0.4%                                    300 xg #1                                                                               5,900,000                                                                                80,000,000                                                                             529,000,000                                                                           0.4%                                    300 xg #2                                                                              12,400,000                                                                                90,000,000                                                                             541,000,000                                                                           0.5%                                    200 xg #1                                                                              15,600,000                                                                                90,000,000                                                                             473,000,000                                                                           0.5%                                    200 xg #2                                                                              17,600,000                                                                                90,000,000                                                                             475,000,000                                                                           0.6%                                    ______________________________________                                    

                                      TABLE 5                                     __________________________________________________________________________    Summary of cell counts pre and post processing of high speed Optipress        ®                                                                         device buffy coat preparations with the Modified Syringe Leukocyte            Collection                                                                    Chamber.                                                                      __________________________________________________________________________    Raw Buffy Coat: Pre Chamber Values                                            Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    7-7:1                                                                              --   22,300,000                                                                           6,130,000,000                                                                        1,644,000,000                                                                        62.3                                                                              --                                         7-28:A                                                                             --   30,200,000                                                                           4,850,000,000                                                                        1,506,000,000                                                                        56.2                                                                              --                                         7-28:B                                                                             --   38,600,000                                                                           5,250,000,000                                                                        1,927,000,000                                                                        57.9                                                                              --                                         8-11 --   13,500,000                                                                           7,200,000,000                                                                        573,000,000                                                                          69.6                                                                              --                                              Mean 2.615E + 07                                                                          5.858E + 09                                                                          1.413E + 09                                                                          61.5                                                                              --                                              Std  9.304E + 06                                                                          9.029E + 08                                                                          5.079E + 08                                                                          5.18                                                                              --                                         __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 1500 xg                   Sample                             mis                                        Id   Time WBC/ml RC/ml  PLT/ml HCT Sample                                     __________________________________________________________________________    7.7:1                                                                              7 Minutes                                                                          10,100,000                                                                           260,000,000                                                                          **     1.3 0.75                                       __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 1500 xg                   Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    7-7:1                                                                              10 Min-                                                                            7,200,000                                                                            220,000,000                                                                          **     1.1 0.8                                             ute                                                                      __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 1000 xg                   Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    7-7:1                                                                              5 Minutes                                                                          53,600,000                                                                           690,000,000                                                                          **     5.1 0.40                                       7-28:A                                                                             5 Minutes                                                                          41,200,000                                                                           340,000,000                                                                          **     3.0 0.56                                       7-28:B                                                                             5 Minutes                                                                          67,400,000                                                                           480,000,000                                                                          **     2.8 0.30                                            Mean 5.407E + 07                                                                          5.033E + 08                                                                          **     3.63                                                                              0.45                                            Std  1.070E + 07                                                                          1.438E + 08                                                                          **     1.04                                                                              0.15                                       7-7:1                                                                              7 Minutes                                                                          6,700,000                                                                            30,000,000                                                                           467,000,000                                                                          0.1 0.40                                       7-28:A                                                                             7 Minutes                                                                          30,500,000                                                                           310,000,000                                                                          **     2.9 0.81                                       7-28:B                                                                             7 Minutes                                                                          74,400,000                                                                           680,000,000                                                                          **     4.0 0.40                                       8-11:                                                                              7 Minutes                                                                          5,700,000                                                                            390,000,000                                                                          2,367,000,000                                                                        1.8 0.65                                            Mean 3.687E + 07                                                                          4.600E+08                                                                            **     2.90                                                                              0.62                                            Std  2.841E + 07                                                                          1.590E+08                                                                            **     0.90                                                                              0.17                                       7-7:1                                                                              10 Min-                                                                            29,400,000                                                                           330,000,000                                                                          **     2.7 0.55                                            ute                                                                      7-28:A                                                                             10 Min-                                                                            20,800,000                                                                           290,000,000                                                                          **     1.4 0.85                                            ute                                                                      7-28:B                                                                             10 Min-                                                                            34,600,000                                                                           620,000,000                                                                          **     3.2 0.50                                            ute                                                                      8-11:                                                                              10 Min-                                                                            5,100,000                                                                            300,000,000                                                                          1,662,000,000                                                                        2.1 0.60                                            ute                                                                           Mean 2.248E + 07                                                                          3.850E + 08                                                                          **     2.35                                                                              0.63                                            Std  1.118E + 07                                                                          1.365E + 08                                                                          **     0.67                                                                              0.13                                       __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Summary of cell counts pre and post processing of high speed                  Optipress ® device buffy coat preparations with the Modified              Syringe Leukocyte Collection Chamber.                                         __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 750 xg                                                       mis                                        Sample                             Sam                                        Id   Time WBC/ml RBC/ml PLT/ml HCT ple                                        __________________________________________________________________________    7-28:A                                                                             5 Minutes                                                                          34,100,000                                                                           290,000,000                                                                          **     1.7 0.50                                       7-28:B                                                                             5 Minutes                                                                          58,600,000                                                                           480,000,000                                                                          3,412,000,000                                                                        2.4 0.30                                            Mean 4.635E + 07                                                                          3.850E + 08                                                                          **     2.05                                                                              0.40                                            Std  1.225E + 07                                                                          9.500E + 07                                                                          **     0.35                                                                              0.10                                       7-7:1                                                                              7 Minutes                                                                          20,900,000                                                                           60,000,000                                                                           985,000,000                                                                          0.3 0.35                                       7-28:A                                                                             7 Minutes                                                                          39,100,000                                                                           310,000,000                                                                          **     3.1 0.65                                       7-28:B                                                                             7 Minutes                                                                          47,600,000                                                                           420,000,000                                                                          2,648,000,000                                                                        2.2 0.40                                       8-11:                                                                              7 Minutes                                                                          1,800,000                                                                            390,000,000                                                                          2,181,000,000                                                                        2.9 0.57                                            Mean 2.735E + 07                                                                          2.950E + 08                                                                          **     2.13                                                                              0.49                                            Std  1.762E + 07                                                                          1.415E + 08                                                                          **     1.11                                                                              0.12                                       7-7:1                                                                              10 Min-                                                                            5,900,000                                                                            30,000,000                                                                           615,000,000                                                                          0.1 0.37                                            ute                                                                      7-28:A                                                                             10 Min-                                                                            32,900,000                                                                           330,000,000                                                                          **     3.0 0.80                                            ute                                                                      7-28:B                                                                             10 Min-                                                                            34,800,000                                                                           440,000,000                                                                          2,778,000,000                                                                        2.2 0.50                                            ute                                                                      8-11:                                                                              10 Min-                                                                            3,000,000                                                                            20,000,000                                                                           278,000,000                                                                          0.1 0.51                                            ute                                                                           Mean 1.915E + 07                                                                          2.050E + 08                                                                          **     1.35                                                                              0.55                                            Std  1.475E + 07                                                                          1.642E + 08                                                                          **     1.28                                                                              0.16                                       __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 500 xg                    Sample                             mis                                        Id   Time WBC/ml RBC/ml PLT/ml HCT Sample                                     __________________________________________________________________________    7-7:1                                                                              7 Minutes                                                                          4,200,000                                                                            60,000,000                                                                           687,000,000                                                                          0.3 0.35                                       7-7:1                                                                              10 Min-                                                                            4,900,000                                                                            30,000,000                                                                           770,000,000                                                                          0.1 0.37                                            ute                                                                      7-28:A                                                                             10 Min-                                                                            34,000,000                                                                           360,000,000                                                                          **     1.7 0.90                                            ute                                                                      7-28:B                                                                             10 Min-                                                                            52,800,000                                                                           5,800,000                                                                            **     3.2 0.40                                            ute                                                                           Mean 3.057E + 07                                                                          1.319E+08                                                                            **     1.67                                                                              0.56                                            Std  1.971E + 07                                                                          1.616E+08                                                                            **     1.27                                                                              0.24                                       __________________________________________________________________________    Post Modified Syringe Leukocyte Collection Chamber: 400 xg                    Sample                             Sample                                     Id   Time WBC/ml RBC/ml PLT/ml HCT Vol.                                       __________________________________________________________________________    7-7:1                                                                              10 Min-                                                                            500,000                                                                              10,000,000                                                                           229,000,000                                                                          0.1 0.20                                            ute                                                                      __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Summary of phenotyping results pre and post processing of high speed          Optipress ® device buffy coat preparations with the modified syringe      leukocyte collection chamber.                                                 Raw Buffy                                                                     Coat                                                                          Pre Chamber  Phenotype of cells expressed %                                   Values       MAb + per HLe + cells                                            Sam-                       Lym-                                               ple                        phocyte                                            ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-           41.10%  26.61%                                                                              17.33%                                                                              6.34% --    5.66%                            7:1                                                                           7-           32.01%  19.46%                                                                              19.07%                                                                              --    14.16%                                                                              6.78%                            28:A                                                                          7-           36.00%  22.65%                                                                              11.61%                                                                              --    6.82% 8.40%                            28:B                                                                          8:11         28.77%  17.97%                                                                              14.87%                                                                              --    23.82%                                                                              4.07%                                 Mean    34.47%  21.67%                                                                              15.72%                                                                              6.34% 11.20%                                                                              6.23%                                 Std     4.61%   3.31% 2.80% NA    8.84% 1.58%                            ______________________________________                                        Raw Buffy Coat: Phenotype of cells expressed % MAb + per HLe                  Pre Chamber Values                                                                            + cells.                                                                      Monocytes   Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time    CD14        CD16    Cr3                                       ______________________________________                                        7-7:1           7.37%       35.29%  --                                        7-28:A          10.25%      --      41.01%                                    7-28:B          17.43%      --      48.16%                                    8-11            8.77%       --      26.55%                                            Mean    10.96%      35.29%  38.57%                                            Std     3.87%       NA      16.43%                                    ______________________________________                                        Post Modi-                                                                    fied                                                                          Syringe                                                                       Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             1500 xg      per HLe + cells                                                  Sam-                                                                          ple                        Lympho-                                            ID   Time    CD3     CD4   cytes  CD16  Cr3  CD19                             ______________________________________                                        7-7:1                                                                              7 Min-  52.78%  34.69%                                                                              23.09% 11.66%     10.79%                                utes                                                                     ______________________________________                                        Post Modified Syringe                                                         Collection       Phenotype of cells expressed % MAb +                         Chamber: 1500 xg per HLe + cells                                                                   Monocytes                                                                              Granulocytes                                                                          Granulocytes                            Sample ID                                                                             Time         CD14     CD16    Cr3                                     ______________________________________                                        7-7:1   7 Minutes                                                                              10.69%     6.92%   --                                        ______________________________________                                        Post Modi-                                                                    fied                                                                          Syringe                                                                       Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             1500 xg      per HLe + cells                                                                             Lym-                                               Sam-                             pho-                                         le                               cytes                                        ID   Time    CD3     CD4   CD8   CD16  Cr3  CD19                              ______________________________________                                        7-7:1                                                                              10 Min- 39.74%  27.22%                                                                              18.12%                                                                              8.09% --   7.29%                                  utes                                                                     ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells                                           Chamber: 1500 xg per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                            Time      CD14       CD16    Cr3                                       ______________________________________                                        7-7:1  10 Minutes                                                                              8.33%      30.14   --                                        ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             1000 xg      per HLe + cells                                                                             Lym-                                               Sam-                       pho-                                               ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-7:1                                                                              5 Min.  51.99%  32.65%                                                                              22.90%                                                                              12.07%                                                                              --    10.03%                           7-   5 Min.  31.34%  18.65%                                                                              9.84% --    12.73%                                                                              15.54%                           28:B                                                                          7-   5 Min.  LS      21.52%                                                                              22.00%                                                                              --    12.07%                                                                              17.20%                           28:A Mean    41.67%  24.27%                                                                              1825% 12.07%                                                                              12.40%                                                                              14.26%                                Std     10.33%  6.04% 13.37%                                                                              NA    6.65% 3.06%                            ______________________________________                                        Post Modified Syringe                                                         Collection       Phenotype of cells expressed % MAb +                         Chamber: 1000xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time     CD14       CD16    Cr3                                       ______________________________________                                        7-7:1   5 Minutes                                                                              11.83%     13.19%  --                                        7-28:B  5 Minutes                                                                              LS         --      5.88%                                     7-28:A  5 Minutes                                                                              14.51%     --      16.72%                                            Mean     13.17%     13.19%  11.30%                                            Std      1.34%      NA      5.42%                                     ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             1000 xg      per HLe + cells                                                                             Lym-                                               Sam-                       pho                                                ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-7:1                                                                              7 Min.  44.39%  25.23%                                                                              18.47%                                                                              12.47%                                                                              --    10.86%                           7-   7 Min.  26.27%  16.26%                                                                              18.24%                                                                              --    22.47%                                                                              24.77%                           28:B                                                                          7-   7 Min.  36.92%  21.61%                                                                              12.23%                                                                              --    17.71%                                                                              16.68%                           28:A                                                                          8-11 7 Min.  29.37%  18.93%                                                                              22.09%                                                                              --    29.40%                                                                              5.95%                                 Mean    34.24%  20.51%                                                                              17.76%                                                                              12.47%                                                                              23.19%                                                                              14.57%                                Std     7.03%   3.32% 3.54% NA    4.80% 7.01%                            ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 1000 xg per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time     CD14       CD16    Cr3                                       ______________________________________                                        7-7:1   7 Minutes                                                                              24.73%     4.89%   --                                        7-28:B  7 Minutes                                                                              20.40%     --      17.19%                                    7-28:A  7 Minutes                                                                              27.27%     --      31.27%                                    8-11    7 Minutes                                                                              11.00%     --      29.12%                                            Mean     20.85%     4.89%   26.86%                                            Std      6.19%      NA      6.19%                                     ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             1000 xg      per HLe + cells                                                                             Lym-                                               Sam-                       pho                                                ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-7:1                                                                              10 Min  7.58%   25.23%                                                                              19.43%                                                                              17.35%                                                                              --    21.07%                           7-   10 Min  11.53%  8.20% 13.98%                                                                              --    22.34%                                                                              45.79%                           28:A                                                                          7-   10 Min  24.22%  21.58%                                                                              12.55%                                                                              --    7.31% 22.19%                           28:B                                                                          8-11 10 Min  28.14%  19.82%                                                                              22.34%                                                                              --    31.25%                                                                              10.34%                                Mean    17.87%  18.71%                                                                              17.08%                                                                              17.35%                                                                              20.30%                                                                              24.85%                                Std     8.54%   5.37% 3.98% NA    9.88% 12.95%                           ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 1000 xg per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                            Time      CD14       CD16    Cr3                                       ______________________________________                                        7-7:1  10 Minutes                                                                              7.07%      6.63%   --                                        7-28:A 10 Minutes                                                                              4.02%      --      8.57%                                     7-28:B 10 Minutes                                                                              28.69%     --      23.10%                                    8-11   10 Minutes                                                                              12.45%     --      26.13%                                           Mean      13.06%     6.63%   19.27%                                           Std       9.52%      NA      7.66%                                     ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype cells expressed % MAb +                                750 xg       per HLe + cells                                                                             Lym-                                               Sam-                       pho-                                               ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-   5 Min.  31.32%  20.42%                                                                              20.16%                                                                              --    22.59%                                                                              28.08%                           28:A                                                                          7-   5 Min.  22.37%  15.15%                                                                              9.34% --    13.43%                                                                              12.29%                           28:B                                                                          Mean 26.85%  17.79%  14.75%                                                                              0.00% 18.01%                                                                              20.19%                                 Std  4.48%   2.64%   5.41% NA    4.58% 7.90%                                  ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 750 xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time     CD14       CD16    Cr3                                       ______________________________________                                        7-28:A  5 Minutes                                                                              15.18%     --      1.11%                                     7-28:B  5 Minutes                                                                              38.33%     --      9.20%                                             Mean     26.76%     0.00%   5.16%                                             Std      11.58%     NA      4.05%                                     ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:      Phenotype of cells expressed % MAb +                            750 xg        per HLe + cells                                                                             Lym-                                              Sam-                        pho-                                              ple                         cytes                                             ID    Time    CD3     CD4   CD8   CD16 Cr3   CD19                             ______________________________________                                        7-7:1 7 Min.  51.86%  25.60%                                                                              23.08%                                                                              LS   --    10.33%                           7-    7 Min.  25.42%  14.62%                                                                              15.19%                                                                              --   20.64%                                                                              LS                               28:A                                                                          7-    7 Min.  24.71%  14.00%                                                                              9.60% --   16.82%                                                                              18.76%                           28:B                                                                          8-11  7 Min.  24.77%  14.48%                                                                              21.18%                                                                              --   39.78%                                                                              11.15%                                 Mean    31.69%  17.23%                                                                              17.26%                                                                              --   25.75%                                                                              13.41%                                 Std     11.65%  4.96% 5.30% NA   10.04%                                                                              3.80%                            ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 750 xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time     CD14       CD16    Cr3                                       ______________________________________                                        7-7:1   7 Minutes                                                                              21.10%     LS      --                                        7-28:B  7 Minutes                                                                              10.22%     --      5.29%                                     7-28:A  7 Minutes                                                                              36.47%     --      6.70%                                     8-11    7 Minutes                                                                              13.44%     --      10.44%                                            Mean     20.31%     --      7.48%                                             Std      10.13%     NA      2.17%                                     ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype of cells expressed % MAb +                             750 xg       per HLe + cells                                                                             Lym-                                               Sam-                       pho-                                               ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-7:1                                                                              10 Min  50.07%  26.74%                                                                              22.64%                                                                              14.67%                                                                              --    13.31%                           7-   10 Min  26.21%  6.22% 16.48%                                                                              --    11.60%                                                                              10.12%                           28:A                                                                          7-   10 Min  23.06%  14.42%                                                                              7.17% --    22.44%                                                                              52.87%                           28:B                                                                          8-11 10 Min  20.85%  15.26%                                                                              25.60%                                                                              --    42.16%                                                                              11.52%                                Mean    30.05%  15.66%                                                                              17.97%                                                                              14.67%                                                                              25.49%                                                                              21.96%                                Std     11.72%  7.31% 7.05% NA    12.65%                                                                              17.88%                           ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 750 xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                            Time      CD14       CD16    Cr3                                       ______________________________________                                        7-7:1  10 Minutes                                                                              19.05%     4.48%   --                                        7-28:A 10 Minutes                                                                              9.19%      --      37.16%                                    7-28:B 10 Minutes                                                                              7.32%      --      3.75%                                     8-11   10 Minutes                                                                              23.06%     --      8.60%                                            Mean      14.66%     4.48%   16.50%                                           Std       6.59%      NA      14.74%                                    ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:       Phenotype of cells expressed % MAb +                           500 xg         per HLe + cells                                                                            Lympho-                                           Sample cytes                                                                  ID     Time    CD3    CD4   CD8    CD16 Cr3  CD19                             ______________________________________                                        7-7:1  7 Min   LS     23.35%                                                                              20.84% LS   --   LS                               ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 500 xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                             Time     CD14       CD16    Cr3                                       ______________________________________                                        7-7-1   7 Minutes                                                                              15.05%     LS      --                                        ______________________________________                                        Post Modi-                                                                    fied Syringe                                                                  Collection                                                                    Chamber:     Phenotype of Cells expressed % MAb +                             500 xg       per HLe + cells                                                                             Lym-                                               Sam-                       pho-                                               ple                        cytes                                              ID   Time    CD3     CD4   CD8   CD16  Cr3   CD19                             ______________________________________                                        7-7:1                                                                              10 Min  38.17%  22.88%                                                                              18.66%                                                                              9.85% --    9.02%                            7-   10 Min  29.98%  16.53%                                                                              19.07%                                                                              --    19.05%                                                                              13.65%                           28:A                                                                          7-   10 Min  25.02%  13.74%                                                                              12.81%                                                                              --    11.94%                                                                              11.87%                           28:B                                                                               Mean    31.06%  17.72%                                                                              16.85%                                                                              9.85% 16.50%                                                                              11.51%                                Std     5.42%   3.82% 2.86% NA    3.56% 1.91%                            ______________________________________                                        Post Modified                                                                 Syringe Collection                                                                             Phenotype of cells expressed % MAb +                         Chamber: 500 xg  per HLe + cells                                                               Monocytes  Granulocytes                                                                          Granulocytes                              Sample ID                                                                            Time      CD14       CD16    Cr3                                       ______________________________________                                        7-7:1  10 Minutes                                                                              25.93%     13.54%  --                                        7-28:A 10 Minutes                                                                              12.63%     --      14.06%                                    7-28:B 10 Minutes                                                                              36.28%     --      10.30%                                           Mean      24.95%     13.54%  12.18%                                           Std       9.68%      NA      1.88%                                     ______________________________________                                    

FIG. 11 illustrates schematically another embodiment of the presentinvention that can be used for purposes of validating virusdepletion/inactivation processes. The method consists of adding virus(e.g., HIV) to a blood component and then treating the blood with aviral inactivation/removal process. Of course, naturally infected bloodcan be used if desired.

Next, virus host cells are added to the blood (e.g., H9 cells for HIV orVero cells for VSV). The cells are allowed to incubate in the wholevolume of the blood component. During this incubation step, that willlast from 1 to 18 hours, infectious virus present in the blood componentattach to and penetrate the host cells to initiate the infectionprocess.

In a further step, pursuant to the present invention, the virus hostcells are harvested and separated from the blood cells by use of aleukocyte filter. The filter removes cell lines used for viralinfectivity studies. Additionally, the filter will remove and separatethe leukocytes from platelets and red cells.

The filter is then washed with tissue culture media, such as 10% FetalCalf Serum in RPMI 1640 media, as required to support the viral hostcells. The filter is then incubated at 37° C. for 6-120 hours to allow avirus to continue propagating in the host cells contained in the filter.

At the end of the incubation period, an effluent is prepared from thefilter (with or without lysing the host cells) to be used as the testmaterial. The effluent is prepared by flushing the filter with a lysisbuffer such as 1% Triton X-100 (Sigma Chemical Co.); 10 mM Tris-HCL, pH7.0; 1 mM EDTA or a hypotonic solution that lysis the leukocytes butdoes not inactivate the virus.

The test material is then either tested for infectious virus usingconventional tissue culture infectivity assays (e.g., plaque-formingunits) or for viral nucleic acid markers (e.g., HIV-DNA sequences usingPCR) or viral antigens (e.g., HIV p24 antigen).

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its attendant advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

We claim:
 1. A method for creating a leukocyte rich sample from a mixedpopulation of blood cells comprising the steps of:separating an amountof blood into layers that include a buffy coat layer having a mixedpopulation of blood cells including plasma and red blood cells;centrifuging the buffy coat layer for a time period between 5 and 10minutes and at a force between 500×g and 1000×g and at a centrifugespeed of less than 3000 rpm, to separate the buffy coat layer into atleast a plasma containing layer, a leukocyte containing layer, and a redblood cell containing layer; and separating the leukocyte containinglayer from the other layers.
 2. The method of claim 1 wherein theleukocyte containing layer includes less than or equal to 5×10⁸ redblood cells.
 3. The method of claim 1 wherein the leukocyte containinglayer includes less than or equal to 5×10⁹ platelets.
 4. The method ofclaim 1 wherein the buffy coat layer is centrifuged in a chamber.
 5. Themethod of claim 1 wherein the step of separating the amount of bloodinto layers includes a centrifugation step.
 6. The method of claim 1wherein the step of separating the amount of blood includes spinning acontainer containing the amount of blood at a speed of less than orequal to 3000 rpm.
 7. The method of claim 1 wherein the step ofseparating the amount of blood includes spinning a container containingthe amount of blood at a speed of at least 3500 rpm.
 8. The method ofclaim 1 wherein the buffy coat layer is centrifuged no longer than 24hours after being collected from a donor.
 9. The method of claim 1wherein the buffy coat layer is centrifuged no longer than 4 hours afterbeing collected from a donor.
 10. A method for creating a leukocyte richsample from a mixed population of blood cells comprising the stepsof:separating an amount of blood into layers that include a buffy coatlayer having a mixed population of blood cells including plasma and redblood cells; centrifuging the buffy coat layer at a centrifugation speedof less than 3000 rpm and at a force of about 750×g to separate thebuffy coat layer into at least a plasma containing layer, a leukocytecontaining layer, and a red blood cell containing layer; separating theleukocyte containing layer from the other layers; wherein the step ofcentrifuging the buffy coat layer occurs within 4 hours after the bloodis collected from a donor.
 11. The method of claim 10 wherein the buffycoat layer is centrifuged for approximately 5 to about 10 minutes.
 12. Amethod for creating a leukocyte rich sample from a mixed population ofblood cells comprising the steps of:separating an amount of blood intolayers that include a buffy coat layer having a mixed population ofblood cells including plasma and red blood cells; centrifuging the buffycoat layer, using a centrifuge speed of less than 3000 rpm and a forceof at least 750×g for a time period between 5 and 10 minutes, toseparate the buffy coat layer into at least a plasma containing layer, aleukocyte containing layer, and a red blood cell containing layer; andseparating the leukocyte containing layer from the other layers.
 13. Themethod of claim 12 wherein the buffy coat layer is centrifuged no longerthan 24 hours after being collected from a donor.
 14. The method ofclaim 12 wherein the buffy coat layer is centrifuged no longer than 4hours after being collected from a donor.